5613327 KNOCK-DOWN OF AUTOPHAGY-RELATED GENE 5 (ATG5) EXPRESSION IN THALASSEMIC CD34+ CELLS LEADS TO AMELIORATION OF ERYTHROPOIESIS
Authors: A. Psychogiou, E. Tsempera, E. Kalafati, E. Voskaridou, E. Drakopoulou, N.P. Anagnou
Journal: HemaSphere (2023)

Background: β-thalassemia is characterized by ineffective erythropoiesis due to increased apoptosis of the thalassemic erythroid precursors. Recent studies documented enhanced initiation of autophagy in β-thalassemia erythroblasts, which may contribute to the elevated levels of apoptosis.1,2 Therefore, genetic manipulation of key genes of autophagy employing shRNA technology, could improve the pathophysiology of the disease. Aim: To this end, our study aimed to investigate the role of ATG5 in autophagy and/or apoptosis in β-thalassemia, by downregulating its expression via shRNA technology, and potentially exploit this effect for the amelioration of the disease pathophysiology. Materials and Methods: Lentiviral vectors (LVs), harboring three distinct shRNAs (sh11840, sh11841 and sh11842) targeting discrete regions of endogenous ATG5 mRNA, were used to transduce K562 cells at a multiplicity of infection (MOI) 25. A lentiviral vector carrying a shRNA against no human gene (shNT; No Target), was used as a control. Flow cytometry and confocal microscopy using an ATG5-specific antibody were employed to determine ATG5 expression at the protein level, while qPCR assessed ATG5 knock-down at the mRNA level. The sh11842 LV exhibited the highest ATG5 knock-down and was selected for in vitro assessment in thalassemic cells, following initial transduction of CD34+ cells at MOI 25, for a differentiation time period to include the early autophagy activation at basophilic stage. Autophagic flux in untransduced, shNT-transduced and shATG5-transduced CD34+ cells, was assessed using confocal microscopy to determine the level of the LC3/LAMP co-localization, markers of autophagosomes and lysosomes, while flow cytometry was employed to determine erythroid differentiation and apoptosis in the differentiated cells. Results: Transduction of K562 cells with the sh11842 LV led to the highest ATG5 knock-down, reaching 93% and 38% at mRNA and protein level, respectively (n=4). Transduction of thalassemic CD34+ cells with the sh11842 LV at day 6 of differentiation showed advanced erythropoiesis compared to shNT-transduced cells. Specifically, the percentage of basophilic erythroblasts was significantly reduced following sh11842-transduction as compared to shNT-transduction, (43.5 ± 4.5% vs 59.6 ± 8.1%, p=0.0127, n=4, respectively), leading to a transient increase of 28.9 ± 5.9% in polychromatic erythroblasts, compared to 16.3 ± 3.7% (p=0.0118, n=4) in shNT-transduced cells. As a result of this accelerated erythropoiesis, the percentage of orthochromatic erythroblasts increased to 5.5 ± 2.4%, compared to 2.8 ± 1.7% observed in shNT-transduced cells (p=0.199, n=4). This was followed by a significant reduction of LC3/LAMP co-localization in sh11842-transduced cells, reaching a Pearson’s correlation coefficient of 0.103 ± 0.29%, compared to 0.165 ± 0.4% seen in shNT-transduced and 0.372 in untransduced cells (p<0.0001). Furthermore, no differences in apoptosis were observed between sh11842- and shNT- transduced cells (59.0 ± 18.2% vs 50.3 ± 8.6%, p=0.6618 n=3). Pilot studies indicate that transduction with sh11842 LV may lead to increased mitochondrial membrane potential, as demonstrated by increased Mitotracker® staining, suggesting a potential reduction in mitophagy, as well. Summary - Conclusion: Knock-down of ATG5 expression leads to improvement of the thalassemic erythropoiesis in vitro, highlighting its potential synergistic role in the amelioration of thalassemic pathophysiology. References 1. Lithanatudom et al, Ann Hematol 2011; 90:747–758 2. Drakopoulou et al, Hum Gene Ther 2019; 30(5):601-617